The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus.. Based on the fact that NS1 protein in infected cell cultures, as well
Trang 1Open Access
Research
Use of a commercial enzyme immunoassay to monitor dengue
virus replication in cultured cells
Juan E Ludert, Clemente Mosso, Ivonne Ceballos-Olvera and Rosa M del
Angel*
Address: Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N., Mexico City, Mexico
Email: Juan E Ludert - jludert@cinvestav.mx; Clemente Mosso - clemen_elche@hotmail.com; Ivonne
Ceballos-Olvera - ivonneceol@yahoo.com.mx; Rosa M del Angel* - rmangel@cinvestav.mx
* Corresponding author
Abstract
Current methods for dengue virus quantitation are either time consuming, technically demanding
or costly As an alternative, the commercial enzyme immunoassay Platelia™ Dengue NS1 AG
(Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells
The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected
with dengue virus The amount of NS1 detected in the supernatants of infected cells was
proportional to the initial MOI used and to the time of post infection harvest This immunoassay
was also able to detect the presence of NS1 in the supernatants of infected human macrophages
Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was
readily monitored with the use of this assay These results suggest that the Platelia™ Dengue NS1
AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue
virus replication in cultured cells
Background
Dengue is one of the most important arthropod-borne
viral diseases in tropical and subtropical areas around the
world and represents a serious public health in several
countries of America, Asia and Africa Last year, only in
the Americas more than 800,000 cases of dengue fever,
the less severe clinical form of dengue infection, and more
than 25,000 cases of dengue hemorrhagic fever, the most
severe form of dengue syndrome, occurred [1] Although
during the past years, the incidence of dengue has grown
in endemic areas, a specific treatment or vaccines are not
yet available
The four antigenically related serotypes of dengue virus
(DEN): DEN1, DEN2, DEN3 and DEN4, members of the
Flavivirus genus (family Flaviviridae), are transmitted to
humans by Aedes aegypti mosquitoes DEN is an
envel-oped virus of 50 nm in diameter and contains a single strand and positive-polarity RNA as genome of about 10.7
kb [2] DEN genome encodes for three structural proteins (envelope glycoprotein, E; membrane, M; and capsid, C) and for seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) E protein is the major struc-tural protein exposed on the surface of the particle
The detection of antibodies directed against E protein is the most common technique to detect DEN infection in diagnostic test, such as the capture enzyme-linked immu-noadsorbent assay MAC-ELISA In addition, some other non-serological techniques such as virus isolation and
Published: 25 April 2008
Virology Journal 2008, 5:51 doi:10.1186/1743-422X-5-51
Received: 12 February 2008 Accepted: 25 April 2008 This article is available from: http://www.virologyj.com/content/5/1/51
© 2008 Ludert et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2ing the acute phase of the disease as well as in
superna-tants of infected cells Virus isolation and titration are also
very useful tools for the quantitation of DEN However,
both procedures are expensive and time-consuming [3,4]
On the other hand, real time PCR or competitive RT-PCR
are also very helpful techniques to quantify viral RNA in
human sera as well as in infected cells [5-9] However, for
molecular methodologies, RNA isolation, expensive
rea-gents, specialized equipment and internal controls are
required Pitfalls in the standard techniques used for the
quantitation of DEN have prompted the search of
alterna-tive semiquatitaalterna-tive methods Recently, a flow
cytometry-based assay and a fluorescent focus assay for flavivirus
quantitation have been reported [10,11]
An attractive alternative for the quantitation of viral
infec-tion efficiency is to measure the amount of a particular
viral protein NS1 is a highly conserved nonstructural
glycoprotein of DEN, which exists predominantly in a
dimeric form, which is associated with intracellular and
cell surface membranes [12] Although the precise role of
NS1 protein in the flavivirus life cycle remains unclear,
NS1 dimers have been shown to interact with other
non-structural viral proteins and through this association, with
the viral RNA Then, NS1 protein may be involved in
assembly of the viral replicase complex and its
localiza-tion to cytoplasmic membranes [13-16] NS1 protein is
secreted from infected cells as a soluble, detergent-labile
hexamer [17] Furthermore, it has been demonstrated that
NS1 antigen is present in the sera of acute-phase infected
patients, and in DEN infected cell cultures, and that
super-natant levels of NS1 protein correlate with infectious titers
[18,19] Recently, a commercial enzyme immunoassay,
Platelia™ Dengue NS1 AG (Bio-Rad Laboratories), was
developed for the detection of NS1 antigen in human
serum or plasma This assay has been reported to be useful
for the diagnosis of DEN infection, particularly during the
early-acute-phase [20,21]
Based on the fact that NS1 protein in infected cell cultures,
as well as in humans, is produced and secreted and that
the amount of NS1 protein correlates with the viral
repli-cation efficiency, in this study we evaluated the use of
Platelia™ Dengue NS1 AG as a surrogate method to
mon-itor semiquantitatively DEN replication in cultured cells
Our results indicate that this commercial assay could be a
useful method to monitor differences in DEN replication
under different experimental conditions in a short time
period (less than three hours)
To evaluate the convenience of the Platelia™ Dengue NS1
AG enzyme immunoassay for the quantitation of NS1 protein in the culture medium of infected cells, sub-con-fluent monolayers of Vero and C6/36 HT cells were infected with different MOIs of DEN2 and the amount of NS1 protein released to the culture media was evaluated
up to 48 hours post infection (hpi) for Vero cells and up
to 60 hpi for C6/36 HT cells using Platelia™ Dengue NS1
AG kit In Vero cells, NS1 protein was detected after 12 hpi and reached a plateau at 36 hpi for all three MOIs used (Figure 1A) At 24 hpi, obvious differences in the level of NS1 were observed between the cells infected with MOIs
of 0.1 and 1 However, these differences were not clear between Vero cells infected at a MOI of 1 and 10
When NS1 levels were measured in supernatants from infected C6/36 HT cells, we could observe that the kinetics
at the three MOI used were very similar, but showed a 12
h lag between them At MOI of 3 the production of NS1 protein reached a plateau 24 h after infection, while at MOI of 0.3 the plateau was reached at 36 h after infection, and at the lowest MOI used (0.03) the plateau was reached at 48 h after infection (Figure 1B) Of note, even though three independent experiments were carried out with each cell line, low standard deviations were obtained for most of the points of the curves, indicating high repro-ducibility for the method Moreover, OD readings obtained from samples run in triplicate were quite similar supporting our previous statement
One plausible explanation for the failure to detect clear differences in NS1 levels in the supernatants obtained from Vero cells inoculated with MOIs of 1 and 10, could
be the high sensitivity of the kit and the saturation of the system To address this possibility, the level of NS1 was monitored in 10 μl aliquots of supernatant of infected cells instead of the 50 μl used previously Upon dilution
of the supernatant, a clear correlation between the MOI used to infect the Vero cells and the level of NS1 produced was observed (Figure 1C) At 24 hpi, an obvious differ-ence in the level of NS1 was observed between the cells infected with MOIs of 1 and 10 Furthermore, the kinetics
at the three MOIs used were very similar, but showed a 12
h lag between them resembling the kinetics observed with C6/36 HT cells (Figure 1B) These results suggest that although the system is saturable, dilutions of the superna-tant permit to reveal differences in the NS1 levels among samples Thus, although Platelia™ Dengue NS1 AG was not designed to be a quantitative assay, it is evident that the OD readings obtained from the ELISA could reflect the amount of NS1 protein produced as a function of time
Trang 3Detection of NS1 protein in supernatant media from cells infected with dengue virus
Figure 1
Detection of NS1 protein in supernatant media from cells infected with dengue virus Vero (A) or C3/36 HT cells
(B) were infected with dengue 2 virus at different MOIs At different times post-infection, the media were collected and tested for the presence of NS1 protein with Platelia™ Dengue NS1 Ag kit (C) Ten micro liters of the supernatant media collected from the experiments shown in (A) were mixed with 40 μl of diluent buffer provided with the kit and used to measure NS1 protein levels with Platelia™ Dengue NS1 Ag kit Points in (A) and (B) are mean values ± SD of three independent experi-ments
M.O.I = 0.0
M.O.I = 10 M.O.I = 1.0 M.O.I = 0.1
0 0.5 1 1.5 2 2.5 3 3.5 4
0 12 24 30 36 48
h.p.i.
A
0 0.5 1 1.5 2 2.5 3 3.5 4
0 12 24 36 48 60
M.O.I = 0.0
M.O.I = 3.0 M.O.I = 0.3 M.O.I = 0.03
h.p.i.
B
C
M.O.I=10 M.O.I=1.0 M.O.I=0.1
0 0.5 1 1.5 2 2.5 3 3.5 4
h.p.i.
A 45
Trang 4Dengue NS1 AG and virus yield, the amount of virus
presents in the supernatants of Vero and C6/36 HT cells
infected with the maximum MOI, 10 and 3 respectively,
were titrated by focus forming unit assay Titer obtained
by plaques and focus forming units assays are very similar
[10] In Vero cells, infectious particles were first detected
after 36 hpi and increased one log at 48 hpi (Figure 2A)
For C6/36 HT cells infected with MOI of 3, virus particles
were first detected after 24 hpi, and increased
exponen-tially at 36, 48 and 60 hpi (Figure 2B)
NS1 protein is detectable in the culture media from
infected macrophages
To test the convenience of the Platelia™ Dengue NS1 AG
for the detection of NS1 protein in DEN2 infected primary
cultures, human peripheral blood CD14 cells isolated
from three healthy donors by anti-CD14 antibody affinity
column, were cultured for 7 days in RPMI 1640 medium
to generate mature macrophages Macrophages were
infected with a MOI of 3 and the infection was permitted
for 24 and 48 h Platelia™ Dengue NS1 AG permits to
monitor DEN replication in the supernatant of primary
cultured cells after 48 hrs, although the OD readings
var-ied widely among donors (0.65, 1.66 and 3.15)
Superna-tants collected at 0 and 24 hpi tested negative for NS1
protein
NS1 protein production can be used to determine the
effect of certain compounds or drugs in viral infection
In an effort to prove that the kit could be used to monitor
infection efficiency under experimental conditions, an
infection inhibition assay was performed It has been
described that the fusion mediated by the E protein
requires the low pH present in internal vesicles [22] Thus,
it was expected that compounds such as NH4Cl would
inhibit DEN fusion and infection in C6/36 HT cells and
that such inhibition could be monitored by measuring the
amount of NS1 protein present in 10 μl of the media with
the Platelia™ Dengue NS1 AG kit C6/36 HT cells were
incubated for 1 h with 50 mM of NH4Cl, 1 h before or 10
min after the infection with DEN2 at MOI of 10 The OD
of the untreated infected cells was considered as 100%
and treated samples were referred as a percentage of the
control Figure 3 shows that the production of NS1
pro-tein was inhibited up to 50% when NH4Cl was added 1 h
before infection while a 90% inhibition was observed
when the NH4Cl was added 10 min after infection
Fur-thermore, the differences observed between treated and
untreated cells in the production of NS1 showed a
rela-tionship with differences in the viral titer, measured using
a plaque forming units assay, obtained in the supernatant
cells Titration by plaque assays is considered the gold standard, but additional techniques which include real time RT-PCR, competitive RT-PCR or flow cytometry using anti-E protein antibodies have also been used for that purpose Although all of them are useful methods, they may be expensive, time-consuming and require spe-cialized equipment [3,4,6,8-11] Therefore, we decided to evaluate the detection of DEN NS1 protein with the Plate-lia™ Dengue NS1 AG kit as a surrogate, semiquantitative method, to monitor viral replication efficiency The Plate-lia™ Dengue NS1 AG assay is a one step microplate enzyme immunoassay that has been reported to be sim-ple, rapid, subject to quality assurance and robust [20,21] Although the precise roles of NS1 protein in the flavivirus life cycle remain unclear, its presence correlates with viral replication efficiency [13-16,19] Our results with Vero and C6/36 HT cells confirm that the secretion of NS1 shows a relationship with viral replication Moreover, given the high sensitivity of the kit, when the amount of NS1 in 50 μl of supernatant are enough to saturate the sys-tem, a low level of viral particles, measured by FFU, were detected This observation indicates that Platelia™ Dengue NS1 AG may actually be used to detect dengue virus infec-tions at early times, when levels of mature virions are still too low to be reliable measured by FFU assays The results with the grow curves obtained in DEN infected Vero as well as in C6/36 HT cells using different MOIs clearly sug-gest that Platelia™ Dengue NS1 AG assay can be used as a fast surrogate marker to monitor semiquantitatively viral infection in both cell types This method may also be a good tool to monitor DEN infection in primary culture cells such as macrophages However, relative quantitation may be difficult due to differences in the number of mac-rophages actually differentiated and infected from each donor Furthermore, the amount of NS1 release from infected macrophages may also be affected by individual host cell factors
Interestingly, several reports indicate that DEN NS1 pro-tein is secreted from mammalian cells but not from mos-quito cells [17,18] In contrast, we found high levels of NS1 protein in the supernatant media of infected C6/36
HT after 24 hpi regardless of the MOI used Secretion of NS1 protein to the culture medium from DEN infected C6/36 cells or from insect cells expressing recombinant dengue virus NS1 protein has been observed by others [23,24] It has been proposed that proper processing of N-glycans appears to be essential for the maturation, trans-port and secretion of NS1 protein from infected cells [17] Thus, discrepancies in the detection of NS1 protein in supernatant from C6/36 infected cells most likely obey to
Trang 5Correlation between focus forming units and NS1 levels in Vero (A) and C6/36 HT (B) cells
Figure 2
Correlation between focus forming units and NS1 levels in Vero (A) and C6/36 HT (B) cells Supernatant from
Vero (A) and C6/36 HT cells (B) infected at a MOI of 10 and 3 respectively were collected and used for: measurement of NS1 protein levels with Platelia™ Dengue NS1 Ag kit (open symbols) and for focus forming units (FFU) calculation (solid symbols) Points are mean values of three independent experiments
A
A45
h.p.i.
0 0.5 1 1.5 2 2.5 3 3.5 4
0 0.5 1 1.5 2 2.5 3 3.5 4
B
A45
h.p.i.
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Trang 6heterogeneities in the glycoconjugates biosynthesis
among the C6/36 cell lines employed [17,23]
On the other hand, the experiments performed with
lyso-somotropic drugs, in which inhibition of DEN infection
by the effect of such agents could be easily monitored,
showed as a proof of principle, that Platelia™ Dengue NS1
AG assay could be used as an excellent tool for fast and
reliable relative quantitation of dengue virus replication
in cultured cells Moreover, the reduction in the level of
NS1 protein released from treated cells correlates with a
reduction in plaque forming units detected in these
super-natants by plaque assay, supporting our previous
conclu-sion The lesser inhibitory effect upon the infection
observed when the cells were pretreated with NH4Cl than
when the drug was administered 10 min after the
inocu-lum must likely reflects the capacity of the cells to extrude
quantitative test Given the sensitivity of the test, maxi-mum OD values are often obtained Furthermore, no information of the limit of NS1 amount detected by the kit is provided by the manufacturer Thus, proper quanti-tation will require serial dilutions of the samples and comparison with standard curves derived from titration series of purified NS1 protein corresponding to the DEN serotype being used [18,19] However, for detection of NS1 protein in cell culture media, this limitation can be overcome partially by using diluted amounts of culture media as shown in Figure 1C or by testing serial dilutions
of the supernatant to reach a detection limit (data not shown) The low standard deviations observed in most of the points of the graphics presented clearly indicate that the results are quite reproducible However to ensure comparability, we recommend testing all samples and controls in the same plate, along with the controls included in the kit
In conclusion, our data suggest that the Platelia™ Dengue NS1 AG assay can be use as a surrogate, easy and fast method for the semiquantitation of DEN in cultured cells Reliable levels of NS1 protein for quantitation are usually reached in the cell supernatant after 24 hpi and the analy-sis can be carried out in less than 3 hours In addition, the low background levels obtained with mock infected cells allowed easy discrimination between positive and nega-tive samples However, DEN quantitation continues to pose difficulties and the development of a quantitative test based on NS1 protein detection is highly desirable
Materials and methods
Cells
Vero cells were grown in Dulbecco's modified eagle medium (D-MEM Advanced, Gibco-BRL) supplemented with 8% fetal calf serum (Gibco-BRL), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 50 U/ml of penicillin and 50 μg/ml of streptomycin BHK21 cells were grown in mini-mal essential medium (MEM, Gibco-BRL) supplemented with 10% fetal calf serum (GIBCO-BRL), 2 g/l sodium bicarbonate, 50 U/ml of penicillin and 50 μg/ml of
strep-tomycin C6/36 HT cells (derived from the C6/36 Aedes
albopictus cells but adapted to grow at 34°C) kindly
pro-vided by Dr Goro Kuno, CDC, Puerto Rico, were grown
in MEM, supplemented with 7% fetal calf serum, nones-sential aminoacids, vitamins, 0.370 g/l sodium bicarbo-nate, 50 U/ml of penicillin and 50 μg/ml of streptomycin
at 34°C
Human peripheral blood mononuclear cells (PBMCs) were purified from peripheral blood obtained from
Detection of NS1 protein in supernatant media from cells
treated with lysosomotropic agents
Figure 3
Detection of NS1 protein in supernatant media from
cells treated with lysosomotropic agents C6/36 HT
cells infected with dengue 2 virus at MOI of 10 were either
treated for 1 h, before infection (-1 hpi) or 10 min
post-infec-tion (10 mpi) with 50 mM NH4Cl (open bars) As a control,
non drug treated cells, either mock infected or infected,
were run in parallel (grey bars) At 24 hpi the supernatant
media were collected and tested for the presence of NS1
protein with Platelia™ Dengue NS1 Ag kit Results are
expressed as the percentage of the OD (A450) obtained in
non-drug treated, infected cells Results are mean values ±
SD of three independent experiments The decrease in NS1
production in drug treated cells was statistically significant
compared to the control condition (* p < 0.05) Titers for
each condition obtained by plaque assay and expressed as
PFU/ml are shown on the top of the bars
0
20
40
60
80
100
120
cells
Infected cells
*
*
Trang 7gradient centrifugation Monocytes were purified from
human PBMCs using MACS CD14 microbeads (Miltenyi
Biotec) according to the manufacturer's
recommenda-tions Purity was checked by FACScan analysis by staining
the cells with an anti-CD14 antibody conjugated with
FITC (Sigma-Aldrich) Macrophages were obtained by
incubation of adherent cells in 96 well plates (1 × 105
cells/well) with RPMI 1640 media supplemented with 5%
fetal calf serum for approximately 7 days
Viruses
DEN serotype 2 (DEN2), strain 16681, generously
pro-vided by Dr Richard Kinney (CDC, Fort Collins, CO), was
propagated in suckling mice brain as previously described
[25] Virus titers in mice brain homogenates were
deter-mined by plaque assay
Virus inoculation
Twenty four-well plates were seeded with either Vero or
C6/36 HT cells and incubated at 37°C in 5% CO2
atmos-phere or at 34°C respectively, until confluence was
reached After washing the monolayers once with PBS,
cells were inoculated with DEN at the appropriate
multi-plicity of infection (MOI) in a final volume of 0.2 ml
Given the higher capacity of DEN to infect mosquito cells
compared to Vero cells, C6/36 HT cells were infected at
MOIs of 3, 0.3 and 0.03 while Vero at MOIs of 10, 1 and
0.1 The virus was left to absorb for 1 h After absorption,
the inoculum was removed by aspiration, the monolayers
were washed once with PBS and 1.0 ml of medium was
added At appropriate times post infection, the
superna-tant media were collected and stored at -20°C for virus
titration by focus forming units assay and to monitor the
presence of NS1 protein with the Platelia™ dengue NS1 Ag
kit
Macrophages, grown in 96-well plates, were infected with
DEN2 at MOI of 3, or mock infected, for 1 hour at 37°C
After inoculation, cells were washed twice with medium,
0.1 ml of fresh RPMI 1640 medium added per well and
infection was allowed to proceed for 0, 24 or 48 h at
37°C The supernatant media were stored at -20°C until
tested for the presence of NS1 protein
Plaque assay
Dengue virus titers were determined by plaque assay on
confluent monolayers of BHK-21 cells grown in 24-well
plates and cultured in MEM-supplemented with 10% fetal
calf serum as previously described [26] Briefly, when the
adherent BHK-21 cells reached 80 to 90% confluence 0.25
ml aliquots of mice brain homogenates or cells
superna-tants from dengue virus-infected C6/36 HT cells were
inoculated at ten-fold serial dilutions from 10-1 to 106
After 4 hrs of viral adsorption, the BHK-21 cell
monolay-ers were overlaid with MEM containing 3%
carboxime-thil-cellulose (Sigma), 0.5% fetal calf serum and 2 mM L-glutamine The cultures were incubated at 37°C for six days and then counted for plaque formation after fixation with 10% formalin and staining 0.5% naphtol-blue-black (Sigma)
Immunohistochemical focus assay
Focus forming assays were carried out as described by Payne et al [11] with modifications Confluent monolay-ers of Vero cells grown in 24-well plates were inoculated with 10-fold serial dilutions of supernatant media in a final volume of 0.25 ml Viral absorption was allowed for
1 h at 37°C An overlay of MEM, 5% fetal calf serum and 1% carboxymethyl-cellulose (Sigma-Aldrich Co., St Louis, MO) was added after the inoculum was removed and cell monolayers were washed once with Hank's solu-tion The overlay was removed at 72 hpi, and cells were fixed for 20 min at room temperature with ice-cold abso-lute methanol DEN foci were labeled with a mixture of anti-DEN E (4G2) and pre-M proteins (2H2) Mabs (a kind gift of Dr Ferdinando Liprandi, Instituto Vene-zolano de Investigaciones Científicas, Caracas) and a sec-ondary antibody conjugated to alkaline phosphatase Foci were stained using a combination of
5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt and nitro-blue
tetra-zolium chloride as substrate (BCIP/NBT kit; Invitrogen, Carlsbad, CA) and counted on a light box with the aid of
a 10× magnifying glass
Lysosomotropic drug treatment
To inhibit the infectious entry of DEN2, C6/36 HT cells grown in 12-well plates were either pretreated with NH4Cl (50 mM, final concentration) for 1 h at 34°C before infec-tion or the drug was added 10 min post infecinfec-tion and left for 1 h Cells were infected at a MOI of 10 in a final vol-ume of 0.5 ml for 1 h at 34°C After inoculation, cells were washed twice with PBS to remove unbound virus and once with acid glycine (pH 2.8) to inactivate viruses that failed to enter After 2 additional washes with PBS, 1.0 ml
of medium was added to each well and cells incubated at 34°C for 24 h At this time, the media were collected and titrated by plaque assay and tested for the presence of NS1 protein Mock infected and virus infected, non-drug treated cells, were run in parallel as negative and positive controls respectively Differences in NS1 levels between drug treated and non-treated cells were tested for
signifi-cance by the Student's t test.
PLATELIA™ assay
The Platelia™ Dengue NS1 Ag is a one step sandwich for-mat microplate enzyme immunoassay for the qualitative
or semi-quantitative detection of dengue NS1 antigen in human sera or plasma The test uses murine monoclonal antibodies for capture and revelation The assay was car-ried out following the procedure indicated by the
Trang 8manu-taneously with the conjugate for 90 min at 37°C within
the microplate wells sensitized with monoclonal
anti-body anti-NS1 For particular experiments, only 10 μl of
supernatant media were used After a washing step, the
presence of immune complexes was detected by a color
development reaction using 3, 3', 5, 5'
tetramethylbenzy-dine as a substrate The color development reaction was
stopped after 30 min incubation at room temperature by
the addition of an acid solution Finally, the optical
den-sity (OD) was determined at 450 nm using an automatic
ELISA plate reader (Multiskan EX, Labsystems) Negative,
positive and cut-off control reagents provided with the kit
were run each time for validation of the assay For
simplic-ity, results are expressed directly as OD and not as a ratio
OD sample/OD cut-off as recommended by the
manufac-turers However, there are no differences if the results are
expressed either way
Competing interests
The authors declare that they have no competing interests
Authors' contributions
JEL performed the experiments presented in figures 1 and
2 CM performed the experiments presented in figure 3
IC–O performed the experiments with human
macro-phages JEL and RMDA conceived of the study,
partici-pated in study design and jointly prepared the
manuscript All the authors read and approved the final
manuscript
Acknowledgements
We thank Ferdinando Liprandi and Lorena Gutierrez for their critical
read-ing of the manuscript and Fernando Medina for technical assistance We
also thank Juan José Campos A from Bio-Rad S.A México, who provided
us the PLATELIA™ kits This work was partially supported by the Consejo
Nacional de Ciencia y Tecnología (CONACYT), Mexico, grant number
48893.
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